Presynaptic 5HT transporters (SERT) control the availability of 5HT following release and recycle 5HT for reuse in subsequent release events, thereby contributing to presynaptic 5HT homeostasis. SERTs are targets for the most commonly prescribed antidepressant medications and genetic variation in SERT has been linked to autism, anxiety, major depressive disorder (MOD) and antidepressant response. Mirroring the network of postsynaptic genes that depend on efficient 5HT clearance to dictate 5HT response, a presynaptic network of interacting proteins and cell signaling pathways dictates appropriate SERT surface abundance and catalytic activity. The Blakely laboratory has been a leader in the field of SERT molecular biology and regulation for over a decade originating with the first identification of SERT genes in mouse and man. In Project 3: Signaling Networks Supporting Serotonin Transport, Blakely's team proposes three Aims to more fully elucidate the identity and regulation of the SERT regulatory proteome in vivo, providing new targets and models to enrich our understanding of how 5HT signaling is established and modulated. In Specific Aim I, Blakely will use both candidate and proteomic approaches to illuminate the SERT regulatory network established in platelets, a rich source for SERT in the periphery, followed by biochemical and anatomical validation of co-expression in neurons, and assessment of the stability of the network to activation of SERT regulatory kinases and phosphatases. In Specific Aim II, Blakely proposes the creation and evaluation of transgenic mouse models that limit PKG mediated regulation of SERT, either through constitutive and raph-specific loss of PKG1 or elimination of a key PKG phosphorylation site in SERT. Secondly, Blakely evaluates the activity of 5HT and antidepressants on SERT trafficking and protein associations via studies of mice harboring the lle172Met allele which reduces SSRI and cocaine recognition at SERT without loss of 5HT uptake function. The ultimate goal is to link changes observed in SERT activity in these models to the stability and organization of the SERT proteome identified in Aim I. In Specific Aim I BLJIlakely's team will examine the broader physiological impact of engineered mutations, collaborating with onte Investigators to explore their impact on 5HT homeostasis and SERT activity in vivo, the abundance If'recessing (editing) and signaling of 5HT receptors, and the physiological and behavioral effects, monitored through dialysis and chronoamperometry studies as well as established behavioral techniques. Together, these efforts will more broadly elucidate how SERT regulation and the SERT regulatory proteome establishes proper 5HT clearance capacity and how genetic variation can influence SERT regulation in vivo.